The present invention relates to the use of and methods and compositions for preparing antiviral nucleoside analogues, particularly FTC (2'-deoxy-5-fluoro-3'-thiacytidine) and prodrug analogues of FTC. More particularly, the invention relates to the B-isomers of these compounds and their selective synthesis and use as antiviral agents.
In 1981, documentation began on the disease that became known as Acquired Immune Deficiency Syndrome (AIDS), as well as its forerunner AIDS Related Complex (ARC). Since that time, the World Health Organization (WHO) has confirmed that 300,000 people have been reported to have developed AIDS. Of these, over 150,000 are in the United States.
In 1983, the cause of the disease AIDS was established as a virus named the human immunodeficiency virus type 1 (HIV-1). As of December, 1990, the WHO estimates that the number of people who are infected with the virus is between 8 and 10 million worldwide and of that number, between 1,000,000 and 1,400,000 are in the U.S. Usually, a person infected with the virus will eventually develop AIDS; in all known cases of AIDS the final outcome has always been death.
The disease AIDS is the end result of HIV infection. The virion replication cycle begins with the virion attaching itself to the host human T-4 lymphocyte immune cell through the bonding of a receptor on the surface of the virion's protective coat (gp 120) with a glycoprotein on the lymphocyte cell (CD4). Once attached, the virion fuses with the cell membrane, penetrates into the host cell, and uncoats its RNA. The virion enzyme, reverse transcriptase, directs the process of transcribing the RNA into single stranded DNA. The viral RNA is degraded and a second DNA strand is created. The now double-stranded DNA is integrated into the T-cell genome.
The host cell uses its own RNA polymerase to transcribe the integrated DNA into viral RNA and the viral RNA directs the production of glycoproteins, structural proteins and viral enzymes for the new virion, which assemble with the viral RNA intact. Once all the components are assembled, the virus buds out of the cell. Thus, the number of HIV-1 virions grows while the number of T-4 lymphocytes declines.
There are at least three critical points in the virion's replication cycle which have been identified as targets for antiviral drugs: (1) the initial attachment of the virion to the T-4 lymphocyte (CD4 glycoprotein), (2) the transcription of viral RNA to viral DNA, and (3) the assemblage of the new virions during replication.
It is the inhibition of the virus at the second critical point, the viral RNA to viral DNA transcription process, that has provided the bulk of the therapies used in treating AIDS. This transcription must occur for the virion to replicate because the virion's genes are encoded in RNA. By introducing drugs that block the enzyme, reverse transcriptase, from transcribing viral RNA to viral DNA successfully, HIV-1 replication can be stopped.
After phosphorylation, nucleoside analogues, such as 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxycytidine (DDC), 2',3'-didehydro-3'-deoxythymidine (D4T), 2',3'-dideoxyinosine (DDI), and various 2'-fluoro-derivatives of these nucleosides are relatively effective in halting HIV replication by inhibiting reverse transcription. Another promising anti-AIDS drug is 2'-deoxy-3'-thiacytidine (BCH-189), which contains an oxathiolane ring instead of the sugar moiety in the nucleoside. This invention provides the new antiviral nucleosides, 2'-deoxy-5-fluoro-3'-thiacytidine (FTC) and various prodrug analogues of FTC, which are unexpectedly potent and nontoxic.
AZT is a successful anti-HIV drug because it prevents the nucleotide chain-linking reaction that elongates viral DNA inside the host T-4 lymphocyte cells or other immune system cells such as macrophages. When AZT enters the cell, cellular kinases activate AZT by phosphorylation to AZT triphosphate. AZT triphosphate then competes with natural thymidine nucleotides for the receptor site of HIV reverse transcriptase enzyme. The natural nucleotide possesses two reactive ends, the 5'-triphosphate end which reacts with the growing nucleotide polymer and the 3'-OH group for linking to the next nucleotide. The AZT molecule only contains the first of these. Once associated with the HIV enzyme active site, the AZT azide group terminates viral DNA formation because the azide cannot make the 3',5'-phosphodiester bond with the ribose moiety of the following nucleoside.
AZT's clinical benefits include increased longevity, reduced frequency and severity of opportunistic infections, and increased peripheral CD4 lymphocyte count. Immunosorbent assays for viral p24, an antigen used to track HIV-1 activity, show a significant decrease with use of AZT. However, AZT's benefits must be weighed against the adverse reactions of bone marrow suppression (neutropenia), nausea, myalgia, insomnia, severe headaches, anemia, and seizures. Furthermore, these adverse side effects occur immediately after treatment begins whereas a minimum of six weeks of therapy is necessary to realize AZT's benefits.
Several other nucleotides inhibit HIV reverse transcription as does AZT triphosphate. Initial tests on 3'-deoxy-3'-fluorothymidine show that its antiviral activity is comparable to that of AZT. DDC and D4T have been tested in vitro against AZT in a delayed drug administration study; both were found to be potent inhibitors of HIV replication with activities comparable (D4T) or superior (DDC) to AZT. Both DDC and D4T are in clinical trials. Although DDC is converted to its 5'-triphosphate less efficiently than its natural analogue, 2'-deoxycytidine, the phosphorylated derivative is resistent to both deaminases and phosphorylases. If dosage and side-effect issues can be resolved, these drugs show potential for becoming effective anti-AIDS drugs.
Currently, DDI is used alone or in conjunction with AZT to treat AIDS. However, DDI's side effects include sporadic pancreatitis and peripheral neuropathy. Owing to its toxicity, reduced doses are necessary and this may limit its usefulness as an antiviral therapeutic treatment. In addition, the drug is susceptible to cleavage under acidic conditions.
Recent cell culture tests on BCH-189 have shown that it possesses anti-HIV activity similar to AZT and DDC, but without as much cellular toxicity. However, BCH-189, like DDC, is toxic at a concentration of .ltoreq.10 .mu.M in intact CEM cells as measured by cell growth and by determining the extent of mitochondrial DNA synthesis, thus suggesting that one of the side effects of BCH-189 might be clinical peripheral neuropathy. Furthermore, although BCH-189 is less toxic to bone-marrow cells than AZT, another side effect of BCH-189, like AZT, might be anemia. Thus, there is a need for superior therapeutic agents such as FTC and FTC prodrug analogues that are provided herein. These agents combine high antiviral activity with minimum toxicity for use as inhibitors of replication and infectivity of HIV in vivo.
The commonly-used chemical approaches for synthesizing nucleosides or nucleoside analogues can be classified into two broad categories: (1) those which modify intact nucleosides by altering the carbohydrate, the base, or both and (2) those which modify carbohydrates and incorporate the base, or its synthetic precursor, at a suitable stage in the synthesis. Because FTC substitutes a sulfur for a carbon atom in the carbohydrate ring, only the second approach is applicable. The most important factor in this latter strategy involves delivering the base from the .beta.-face of the carbohydrate ring in the glycosylation reaction because only the .beta.-isomers exhibit useful biological activity.
It is well known in the art that the stereoselective introduction of bases to the anomeric centers of carbohydrates can be controlled by capitalizing on the neighboring group participation of a 2-substituent on the carbohydrate ring [Chem. Ber. 114:1234 (1981)]. However, FTC and its analogues do not possess an exocyclic 2-substituent and, therefore, cannot utilize this procedure unless additional steps to introduce a functional group that is both directing and disposable are incorporated into the synthesis. These added steps would lower the overall efficiency of the synthesis.
It is also well known in the art that "considerable amounts of the undesired .alpha.-nucleosides are always formed during the synthesis of 2'-deoxyribosides" [Chem. Ber. 114:1234, 1244 (1981)]. Furthermore, this reference teaches that the use of simple Friedel-Crafts catalysts like SnCl.sub.4 in nucleoside syntheses produces undesirable emulsions upon the workup of the reaction mixture, generates complex mixtures of the .alpha. and .beta.-isomers, and leads to stable .alpha.-complexes between the SnCl.sub.4 and the more basic silyated heterocycles such as silyated cytosine. These complexes lead to longer reaction times, lower yields, and production of the undesired unnatural N-3-nucleosides. Thus, the prior art teaches the use of trimethysilyl triflate or trimethylsilyl perchlorate as a catalyst during the coupling of pyrimidine bases with a carbohydrate ring to achieve the highest yields of the biologically active .beta.-isomers. However, the use of these catalysts to synthesize FTC or FTC analogues exhibit little preference for the desired .beta.-isomer; these reactions typically result in mixtures containing nearly equal amounts of both isomers. Thus, there exists a need for an efficient synthetic route to FTC and FTC prodrug analogues.